Methods of increasing cerebral blood flow

ABSTRACT

Methods of increasing blood flow in a mammalian brain blood vessel characterized by, or otherwise experiencing, decreased blood flow due to an ischemic or other hypoxic event, vasoconstriction or vasospasm following hemorrhagic stroke; due to chronic high blood pressure; and/or due to idiopathic causes are provided. The method for increasing blood flow in such a mammalian brain blood vessel includes administering to a patient in need thereof a therapeutically effective amount of an inhibitor of δ protein kinase C. In certain embodiments, the inhibitor can be chronically administered without causing desensitization of the patient to the inhibitor. Kits for increasing blood flow in a mammalian brain blood vessel characterized by, or otherwise experiencing, decreased blood flow due to an ischemic or other hypoxic event, vasoconstriction or vasospasm following hemorrhagic stroke; due to chronic high blood pressure; and/or due to idiopathic causes are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/317,806,filed Dec. 23, 2005, now allowed, which claims the benefit of U.S.Provisional Application No. 60/641,413, filed Jan. 4, 2005. Bothdocuments are incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work was supported in part by the National Institutes of Healthunder grant number NS44350. Accordingly, the United States Governmenthas certain rights in the invention.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created 22 Jun. 2009, and named“586008214US01seqlist.txt” (4,584), the contents of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The subject matter described herein relates to methods of increasingcerebral blood flow in a vessel experiencing decreased blood flow. Morespecifically, the subject matter relates to methods of increasingcerebral blood flow in a blood vessel by administering an inhibitor ofdelta protein kinase C (δPKC).

BACKGROUND

An ischemic stroke occurs when the flow of blood to the brain isobstructed. Cerebral ischemia and reperfusion injury, due to diseasessuch as stroke and cardiac arrest, are a leading cause of disability anddeath in the US. Over 700,000 strokes occur annually in the UnitedStates (U.S.) alone, and, as of 2001, 4,800,000 people were living withthe consequences of stroke. The overall death rate for stroke isapproximately 58%, with approximately 50% of these patients dying in ahospital (American Heart Association Heart Disease and StrokeStatistics, 2004 Update).

In addition to ischemic stroke, hemorrhagic stroke (includingintracerebral and subarachnoid types) account for 12% of all strokeincidents. Unlike ischemic strokes, hemorrhagic stroke is due to loss ofblood from the vascular system into the parenchymal, subarachnoid orsubdural space. This type of stroke incurs a much higher mortality rate(37-38% mortality within 30 days) in comparison to ischemic stroke.(American Heart Association Heart Disease and Stroke Statistics, 2004Update). Hemorrhagic stroke often leads to the production of chronicdelayed cerebral vasoconstriction and vasospasm. In fact, ⅔ of patientswhich have been treated for subarachnoid hemorrhage suffer from cerebralvasospasm between days 3 and 13 after hemorrhage. Suhardja, A., NatureClin. Prac., Cardiovasc. Med. 1(2):110-116 (2004). These alterations incerebral vascular function occur both in larger diameter vessels (e.g.,basilar artery, middle cerebral artery), and in smaller diametervessels, including capillaries and arterioles. Vasospasm is due in partto build up of reactive blood products surrounding the vasculature,including oxyhemoglobin, which sequesters the vasodilator nitric oxide(NO). Loss of cerebral blood flow autoregulation, includingvasoconstriction and vasospasm, lead to increased cerebral damage fromrestricted blood flow, causing worsening of brain damage in the areasurrounding the trauma, and accounting in part for the worsened outcomeof hemorrhagic stroke patients.

Other causes of compromised cerebral vascular autoregulation includechronic high blood pressure, or hypertension. Hypertension affectsapproximately 1 in 5 Americans (1 in 4 adults) (American HeartAssociation Heart Disease and Stroke Statistics, 2004 Update) and is asignificant risk factor for cerebral stroke injury, in addition to otherchronic diseases including congestive heart failure. Hypertension hasbeen correlated with reduced cerebral blood flow (Rodriguez, G., et al.,Stroke 18(1):13-20 (1987)). Therefore, maintaining adequate cerebralperfusion is critical in managing cerebral protection in these at-riskpatients. Control of cerebral blood flow is also important formaintaining brain perfusion in other idiopathic causes of cerebralvasoconstriction and vasospasm, including migraine, pregnancy,Call-Fleming syndrome and benign angiopathy of the central nervoussystem (Singhal, A. B. Top. Stroke Rehabil. 11(2),1-6 (2004)).

Additionally, the cost of cerebrovascular disease is enormous. Both fromdirect health expenditures and lost productivity due to morbidity andmortality, the cost of stroke in the U.S. amounts to approximately $53.6billion. (American Heart Association Heart Disease and StrokeStatistics, 2004 Update).

Despite this large clinical need, current treatment options for cerebralischemic and reperfusion injury are limited. The only clinicallyapproved medication for stroke, recombinant tissue plasminogen activator(rtPA), is only used in about 1% to about 2% of patients nationally, duein large part to a short therapeutic window and high risk profile,including increased risk of cerebral hemorrhage and death.

In designing a drug or treatment for ischemic stroke, it is desirable todesign a drug or treatment that may be administered multiple timeswithout causing patient desensitization. Desensitization of patients toa drug occurs when the drug is administered and either has notherapeutic effect or the therapeutic effect decreases as a function ofthe number of times that the drug is administered. This occurs withseveral medications, including, for example, nitroglycerine used totreat cardiac ischemia.

There is therefore a need for a drug or treatment method for increasingblood flow in the blood vessels of the brain. There is further a needfor such a drug or treatment method that does not cause patientdesensitization, and therefore may allow chronic administration asneeded. The present invention addresses these needs.

SUMMARY

It has been discovered that an inhibitor of delta protein kinase C(δPKC) can increase cerebral blood flow in a mammal experiencingdecreased cerebral blood flow due to an ischemic or other hypoxic event.Accordingly, methods for increasing blood flow in such a mammalian brainblood vessel experiencing decreased blood flow are provided.Furthermore, methods for increasing blood flow in such a mammalian bloodvessel that do not cause desensitization are provided. Kits forincreasing blood flow in such a mammalian brain blood vessel are alsoprovided.

In a first aspect of the invention, methods for increasing blood flow ina mammalian brain blood vessel characterized by, or otherwiseexperiencing, decreased blood flow due to a disease or condition, suchas an ischemic event, are provided. In one form, a method for increasingblood flow in such a mammalian brain blood vessel includes administeringto a patient in need thereof a therapeutically effective amount of aninhibitor of δ protein kinase C. In certain forms of the invention, theinhibitor is a peptide, and is further a δV1-1 peptide. In alternateforms of the invention, the inhibitor is a δV1-2 peptide, a δV1-5peptide or a δV5 peptide. Fragments or derivatives of the aforementionedpeptides that may advantageously be used in the methods of the inventionare further described. The methods may advantageously be used toeffectively increase cerebral blood flow in conditions characterized bydecreased cerebral blood flow. For example, the methods may be used toincrease cerebral blood flow, and to thereby reduce injury, from anischemic event, such as an ischemic stroke or from vasoconstriction orvasospasms following a hemorrhagic stroke. The methods may also beutilized to increase blood flow in a brain blood vessel in vivo for anyother purpose where increased blood flow is desired, including inindividuals experiencing decreased blood flow in one or more vessels dueto chronic high blood pressure and/or due to idiopathic causes.

In one form of the invention, a method for increasing blood flow in amammalian brain blood vessel characterized by, or otherwiseexperiencing, decreased blood flow due to a disease or conditiondescribed herein, or otherwise known in the art, includes administeringto a patient in need thereof a therapeutically effective amount of aninhibitor of δ protein kinase C wherein the inhibitor is capable ofchronic administration without causing patient desensitization to theinhibitor.

In yet other forms of the invention, a method for increasing blood flowin a mammalian brain blood vessel characterized by, or otherwiseexperiencing, decreased blood flow due to a disease or conditiondescribed herein and/or otherwise known in the art includesadministering to a patient in need thereof a therapeutically effectiveamount of an inhibitor of δ protein kinase C multiple times withoutcausing desensitization to the inhibitor.

In a second aspect of the invention, kits for increasing blood flow in amammalian brain blood vessel characterized by, or otherwiseexperiencing, decreased blood flow due to a disease or conditiondescribed herein and/or otherwise known to the art are provided. In oneform, a kit includes an inhibitor of δ protein kinase C as describedherein and instructions for using the inhibitor to increase blood flowin one or more cerebral blood vessels.

It is an object of the invention to provide methods for increasing bloodflow in a mammalian brain blood vessel characterized by, or otherwiseexperiencing, decreased blood flow due to a disease or condition, suchas, for example, an ischemic event or vasoconstriction or vasospasmfollowing a hemorrhagic stroke; due to chronic high blood pressure;and/or due to idiopathic causes.

It is a further object of the invention to provide methods forincreasing blood flow in a mammalian brain blood vessel characterizedby, or otherwise experiencing, decreased blood flow due to a disease orcondition described herein and/or otherwise known to the art that do notcause patient desensitization to the drug effect.

It is yet another object of the invention to provide kits for increasingblood flow in a mammalian brain blood vessel characterized by, orotherwise experiencing, decreased blood flow due to a disease orcondition described herein and/or otherwise known to the art.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graph of cerebral blood flow (CBF) as a function of timein sham (non-stroked) rats treated with a therapeutic dosage of δV1-1peptide as more fully described in Example 1. The arrow denotes deliveryof δV1-1 peptide, Sham-treated rats were treated with the δV1-1 peptideand the CBF was measured, all as described in Example 1.

FIG. 2 depicts a graph of CBF as a function of time in rats subjected toischemic/reperfusion injury by middle cerebral artery (MCA) occlusionand treated with a therapeutic dosage of δV1-1 as more fully describedin Example 1. The arrow denotes delivery of δV1-1 peptide.

FIG. 3 shows a graph of CBF as a function of time in rats subjected toischemic/reperfusion injury and treated with repeated administrations ofa therapeutic dosage of δV1-1 as more fully described in Example 2. Thearrows denote delivery of δV1 -1 peptide.

FIGS. 4A-4L are computer-generated photomicrographs of brain tissue fromcontrol (healthy) rats not subjected to ischemic/reperfusion injury(FIGS. 4A, 4D, 4G, 4J) and from rats subjected to ischemic/reperfusioninjury by middle cerebral artery (MCA) occlusion and treated with TATpeptide (FIGS. 4B, 4E, 4H, 4K) or with δV1-1-TAT (FIGS. 4C, 4F, 4I, 4L).

FIG. 5 is a bar graph of the average number of microvascular vessels inthe microscope field for animals subjected to ischemic/reperfusioninjury by middle cerebral artery (MCA) occlusion and treated with TATpeptide or with δV1-1-TAT-treated tissue Sham treated animals weresimilarly treated, but were not subjected to ischemic/reperfusion injuryor delivery of peptides.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the subject matterdescribed herein, reference will now be made to preferred embodimentsand specific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope is therebyintended, such alterations and further modifications of the subjectmatter, and such further applications of the principles illustratedherein, being contemplated as would normally occur to one skilled in theart to which the subject matter relates.

Methods of increasing blood flow in a vertebrate brain blood vesselcharacterized by, or otherwise experiencing, decreased blood flow due toa disease or condition are provided. The disease or condition is onethat causes decreased cerebral blood flow. The decreased blood flow mayarise from a variety of events, including an ischemic stroke,reperfusion, idiopathic causes or vasoconstriction or vasospasm 1)following or otherwise associated with a hemorrhagic stroke; 2) due tochronic high blood pressure; 3) due to reperfusion; and/or 4) due toidiopathic causes. It has been discovered that selected isozymes ofprotein kinase C (PKC) increase the flow of blood in such a mammalianbrain blood vessel. It has also been discovered that such isozymes maybe advantageously utilized to reduce cell, tissue or organ damage, deathor other injury due to conditions characterized by decreased blood flow,including injury due to a hypoxic event, such as an ischemic stroke, orinjury due to vasoconstriction or vasospasm following a hemorrhagicstroke; due to chronic high blood pressure; and/or due to idiopathiccauses. By “hypoxic event” or “hypoxia”, it is meant herein an eventwhich causes a cell, tissue or organ to receive an inadequate supply ofoxygen. “Ischemic stroke”, “ischemia” or “ischemic event”, as usedherein refers to an insufficient supply of blood to a specific cell,tissue or organ. A consequence of decreased blood supply is aninadequate supply of oxygen (i.e., hypoxia) and nutrients to the cell,tissue or organ.

Although not being limited by theory, it is believed that during and/orafter an ischemic event there exists a hypoperfused area of tissue knownas the ischemic penumbra which surrounds the tissue directly fed fromthe occluded vessel, or is in brain areas outside the direct peripheryof the ischemic core. Although some of this tissue area directly fedfrom the occluded vessel, known as the ischemic core, may beirreversibly damaged within minutes of the start of the ischemic event,it is believed herein that the penumbral tissue may be salvaged bymaintaining blood flow and/or vessel patency.

A hypoperfused area can also occur in patients who have vasoconstrictionfollowing hemorrhagic stroke; due to chronic high blood pressure and/ordue to idiopathic causes. It is believed herein that the hypoperfusioncaused by the vasoconstriction induces neuronal, glial and vasculardamage that may be salvaged by improving blood flow and/or vesselpatency. In one form of the invention, a method includes administeringto a patient in need thereof a therapeutically effective amount of aninhibitor of δ protein kinase C (δPKC).

It has also been discovered that the inhibitor of δPKC can bechronically administered without inducing desensitization to theinhibitor. Accordingly, in yet another form of the invention, a methodof increasing blood flow in a vertebrate brain blood vesselcharacterized by, or otherwise experiencing, decreased blood flow due toa disease or condition described herein includes administering to apatient a therapeutically effective amount of an inhibitor of δPKC,wherein the inhibitor is capable of chronic administration withoutcausing desensitization to the inhibitor. Kits for increasing blood flowin a mammalian brain blood vessel characterized by, or otherwiseexperiencing, decreased blood flow due to a disease or conditiondescribed herein are also provided. In one form, a kit includes aninhibitor of δPKC and instructions for using the inhibitor to increaseblood flow in a vertebrate brain blood vessel experiencing decreasedblood flow due to a disease or condition described herein.

In one aspect, methods of increasing blood flow in a vertebrate brainblood vessel characterized by, or otherwise experiencing, decreasedblood flow due to a disease or condition are provided. In oneembodiment, a method includes administering to a patient atherapeutically effective amount of an inhibitor of δPKC.

By “blood flow”, it is meant generally herein the amount of bloodflowing in the respective vessel per unit time, By “increasing bloodflow”, it is meant herein that the blood flow at a particular time isincreased relative to the blood flow at some predetermined time point,such as during the onset of, or a specified time period after the onsetof, an ischemic, hypoxic or other cell, tissue or organ injuring eventdescribed herein and/or known in the art; including when a patient isexperiencing vasoconstriction or vasospasm due to a hemorrhagic strokeor while experiencing chronic high blood pressure. Blood flow istypically increased after treatment with the inhibitors described hereinrelative to the blood flow prior to such treatment. By “decreased bloodflow” it is meant herein that the blood flow at a particular time isdecreased relative to blood flow at some predetermined time point, suchas prior to the onset of, or a specified time period after the onset of,an ischemic, hypoxic or other cell, tissue or organ injuring eventdescribed herein and/or known in the art; including when a patient isexperiencing vasoconstriction or vasospasm due to, for example, ahemorrhagic stroke or while experiencing chronic high blood pressure.Several methods are known in the art for determining blood flow in oneor more blood vessels. For example, Laser Doppler Flowmetry (LDF),Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET),and Computed Tomography (CT) Imaging, including Single Photon EmissionComputed Tomography (SPECT) may be used to determine cerebral blood flow(Leenders, K. L., et al. Brain 113:27-47 (1990); Sakai, F., et al. J.Cereb. Blood Flow Metab. 5:207-213 (1988); Rempp, K. A., et al,Radiology 193:637-641 (1994); Baird, A. E. and Warach, S., J. Cereb.Blood Flow Metab. 18:583-609 (1998); Danus, G., et al., Radiology213:141-149 (1999); Calamante, F., et al., J. Cereb. Blood Flow Metab.19:701-735 (1999); Ginsberg, M. D., et al., J. Cereb Blood Flow Metab.2(1):89-98 (1982); Fukuda, O., Neurosurgery 36(2):358-364 (1995);Perez-Pinzon, et al., J. Neurolog. Sci. 153 (1):25-31 (1997); Borlongan,et al., Brain Res. 1010(1-2):108-116 (2004)). It is realized that theseand other known methods may involve different measurements and may notprovide blood flow in terms of volume per unit time. For example, LDFprovides a value for blood flow that is called the red blood cell flux,defined as the product of the number of red blood cells and theirvelocity. Blood flow as described herein may therefore be reported in awide variety of formats and/or units.

A wide variety of inhibitors of δPKC may be utilized in the describedmethod. By inhibitor of δPKC, it is meant herein a compound thatinhibits the biological activity or function of δPKC. As known in theart, δPKC is involved a myriad of cellular processes, includingregulation of cell growth, and regulation of gene expression. Theinhibitors may, for example, inhibit the enzymatic activity of δPKC. Theinhibitors may inhibit the activity of δPKC by, for example, preventingactivation of δPKC or may prevent binding of δPKC to its proteinsubstrate. Such an inhibition of enzymatic activity would prevent, forexample, phosphorylation of amino acids in proteins. The inhibitor mayalso prevent binding of δPKC to its anchoring protein, also known asreceptor for activated kinase (RACK) and the associated translocation ofδPKC to its subcellular location.

In one form of the method, organic molecule inhibitors, includingalkaloids, may be utilized. For example, benzophenanthridine alkaloidsmay be used, including chelerythrine, sanguirubine, chelirubine,sanguilutine, and chililutine. Such alkaloids can be purchasedcommercially and/or isolated from plants as known in the art and asdescribed, for example, in U.S. Pat. No. 5,133,981.

The bisindolylmaleimide class of compounds may also be used asinhibitors of δPKC. Exemplary bisindolylmaleimides includebisindolylmaleimide I, bisindolylmaleimide II, bisindolylmaleimide III,bisindolylmaleimide IV, bisindolylmaleimide V, bisindolylmaleimide VI,bisindolylmaleimide VI, bisindolylmaleimide VIII, bisindolylmaleimideIX, bisindolylmaleimide X and other bisindolylmaleimides that areeffective in inhibiting δPKC. Such compounds may be purchasedcommercially and/or synthesized by methods known to the skilled artisanand as described, for example, in U.S. Pat. No. 5,559,228 and Brenner,et al., Tetrahedron 44(10) 2887-2892 (1988). Anti-helminthic dyesobtained from the kamala tree and effective in inhibiting δPKC may alsobe utilized, including rottlerin, and may be purchased commercially orsynthesized by the skilled artisan.

In certain forms of the method, a protein inhibitor of δPKC may beutilized. The protein inhibitor may be in the form of a peptide.Protein, peptide and polypeptide as used herein and as known in the artrefer to a compound made up of a chain of amino acid monomers linked bypeptide bonds. Unless otherwise stated, the individual sequence of thepeptide is given in the order from the amino terminus to the carboxylterminus. The protein inhibitor of δPKC may be obtained by methods knownto the skilled artisan. For example, the protein inhibitor may bechemically synthesized using various solid phase synthetic technologiesknown to the art and as described, for example, in Williams, Paul Lloyd,et al. Chemical Approaches to the Synthesis of Peptides and Proteins,CRC Press, Boca Raton, Fla., (1997).

Alternatively, the protein inhibitor may be produced by recombinanttechnology methods as known in the art and as described, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor laboratory, 2^(nd) ed., Cold Springs Harbor, N.Y. (1989), Martin,Robin, Protein Synthesis: Methods and Protocols, Humana Press, Totowa,N.J. (1998) and Current Protocols in Molecular Biology (Ausubel et al.,eds.), John Wiley & Sons, which is regularly and periodically updated.For example, an expression vector may be used to produce the desiredpeptide inhibitor in an appropriate host cell and the product may thenbe isolated by known methods. The expression vector may include, forexample, the nucleotide sequence encoding the desired peptide whereinthe nucleotide sequence is operably linked to a promoter sequence.

As defined herein, a nucleotide sequence is “operably linked” to anothernucleotide sequence when it is placed in a functional relationship withanother nucleotide sequence. For example, if a coding sequence isoperably linked to a promoter sequence, this generally means that thepromoter may promote transcription of the coding sequence. Operablylinked means that the DNA sequences being linked are typicallycontiguous and, where necessary to join two protein coding regions,contiguous and in reading frame. However, since enhancers may functionwhen separated from the promoter by several kilobases and intronicsequences may be of variable length, some nucleotide sequences may beoperably linked but not contiguous. Additionally, as defined herein, anucleotide sequence is intended to refer to a natural or syntheticlinear and sequential array of nucleotides and/or nucleosides, andderivatives thereof. The terms “encoding” and “coding” refer to theprocess by which a nucleotide sequence, through the mechanisms oftranscription and translation, provides the information to a cell fromwhich a series of amino acids can be assembled into a specific aminoacid sequence to produce a polypeptide.

The inhibitor may be derived from an isozyme of PKC, such as δV1-1,whose amino acid sequence from Rattus norvegicus is set forth in SEQ IDNO:1 (SFNSYELGSL), representing amino acids 8-17 of rat δPKC as found inGenbank Accession No. AAH76505. Alternatively, the peptide inhibitor maybe other fragments of PKC, such as δV1-2, δV1-5 and/or δV5, or somecombination of δV1-1, δV1-2, δV1-5 and δV5. The amino acid sequence ofδV1-2 from Rattus norvegicus is set forth in SEQ ID NO:2 (ALTTDRGKTLV),representing amino acids 35 to 45 of rat δPKC found in Genbank AccessionNo. AAH76505. The amino acid sequence of δV1-5 from Rattus norvegicus isset forth in SEQ ID NO:3 (KAEFWLDLQPQAKV), representing amino acids 101to 114 of rat δPKC found in Genbank Accession No. AAH76505. The aminoacid sequence of δV5 is set forth in SEQ ID NO:4, representing aminoacids 569-626 of human δPKC found in Genbank Accession No. BAA01381,with the exception that amino acid 11 (aspartic acid) is substitutedwith a proline.

The peptide inhibitors may include natural amino acids, such as theL-amino acids or non-natural amino acids, such as D-amino acids. Theamino acids in the peptide may be linked by peptide bonds or, inmodified peptides described herein, by non-peptide bonds.

A wide variety of modifications to the amide bonds which link aminoacids may be made and are known in the art. Such modifications arediscussed in general reviews, including in Freidinger, R. M. “Design andSynthesis of Novel Bioactive Peptides and Peptidomimetics” J. Med. Chem.46:5553 (2003), and Ripka, A. S., Rich, D. H. “Peptidomimetic Design”Curr. Opin. Chem. Biol. 2:441 (1998). These modifications are designedto improve the properties of the peptide by increasing the potency ofthe peptide or by increasing the half-life of the peptide.

The potency of the peptide may be increased by restricting theconformational flexibility of the peptide. This may be achieved by, forexample, including the placement of additional alkyl groups on thenitrogen or alpha-carbon of the amide bond, such as the peptoid strategyof Zuckerman et al, and the alpha modifications of, for example Goodman,M. et. al. (Pure Appl. Chem. 68:1303 (1996)). The amide nitrogen andalpha carbon may be linked together to provide additional constraint(Scott et al, Org. Letts. 6:1629-1632 (2004)).

The half-life of the peptide may be increased by introducingnon-degradable moieties to the peptide chain. This may be achieved by,for example, replacement of the amide bond by a urea residue (Patil etal, J. Org. Chem. 68:7274-7280 (2003)) or an aza-peptide link (Zega andUrleb, Acta Chim. Slov. 49:649-662 (2002)). Other examples ofnon-degradable moieties that may be introduced to the peptide chaininclude introduction of an additional carbon (“beta peptides”, Gellman,S. H. Acc. Chem. Res. 31:173 (1998)) or ethene unit (Hagihara et al, J.Am. Chem. Soc. 114:6568 (1992)) to the chain, or the use ofhydroxyethylene moieties (Patani, G. A., Lavoie, E. J. Chem. Rev.96:3147-3176 (1996)) and are also well known in the art. Additionally,one or more amino acids may be replaced by an isosteric moiety such as,for example, the pyrrolinones of Hirschmann et al (J. Am. Chem. Soc.122:11037 (2000)), or tetrahydropyrans (Kulesza, A. et al., Org. Letts.5:1163 (2003)).

Although the peptides are described primarily with reference to aminoacid sequences from Rattus norvegicus, it is understood that thepeptides are not limited to the specific amino acid sequences set forthin SEQ ID NOS:1-4. Skilled artisans will recognize that, through theprocess of mutation and/or evolution, polypeptides of different lengthsand having different constituents, e.g., with amino acid insertions,substitutions, deletions, and the like, may arise that are related to,or sufficiently similar to, a sequence set forth herein by virtue ofamino acid sequence homology and advantageous functionality as describedherein. The terms “δV1-1 peptide”, “δV1-2 peptide”, “δV1-5 peptide” and“δV5 peptide” are used to refer generally to the peptides having thefeatures described herein and preferred examples include peptides havingthe amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQID NO:4, respectively. Also included within this definition, and in thescope of the invention, are variants of the peptides which function inincreasing cerebral blood flow as described herein.

The peptide inhibitors described herein also encompass amino acidsequences similar to the amino acid sequences set forth herein that haveat least about 50% identity thereto and function to increase cerebralblood flow. Preferably, the amino acid sequences of the peptideinhibitors encompassed in the invention have at least about 60%identity, further at least about 70% identity, preferably at least about75% or 80% identity, more preferably at least about 85% or 90% identity,and further preferably at least about 95% identity, to the amino acidsequences, including SEQ ID NOS:1-4, set forth herein.

Percent identity may be determined, for example, by comparing sequenceinformation using the advanced BLAST computer program, including version2.2.9, available from the National Institutes of Health. The BLASTprogram is based on the alignment method of Karlin and Altschul. Proc.Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul,et al., J. Mol. Biol. 215:403-410 (1990); Karlin And Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., NucleicAcids Res. 25:3389-3402 (1997). Briefly, the BLAST program definesidentity as the number of identical aligned symbols (i.e., nucleotidesor amino acids), divided by the total number of symbols in the shorterof the two sequences. The program may be used to determine percentidentity over the entire length of the proteins being compared. Defaultparameters are provided to optimize searches with short query sequencesin, for example, blastp with the program. The program also allows use ofan SEG filter to mask-off segments of the query sequences as determinedby the SEG program of Wootton and Federhen, Computers and Chemistry17:149-163 (1993).

Accordingly, fragments or derivatives of peptide inhibitors describedherein may also be advantageously utilized that include amino acidsequences having the specified percent identities to SEQ ID NOS:1-4described herein to increase cerebral blood flow. For example fragmentsor derivatives of δV1-1, δV1-2, δV1-5 and δV5 that are effective ininhibiting δPKC and increasing cerebral blood flow in a mammal asdescribed herein may also advantageously be utilized in the presentinvention.

Conservative amino acid substitutions may be made in the amino acidsequences described herein to obtain derivatives of the peptides thatmay advantageously be utilized in the present invention. Conservativeamino acid substitutions, as known in the art and as referred to herein,involve substituting amino acids in a protein with amino acids havingsimilar side chains in terms of, for example, structure, size and/orchemical properties. For example, the amino acids within each of thefollowing groups may be interchanged with other amino acids in the samegroup: amino acids having aliphatic side chains, including glycine,alanine, valine, leucine and isoleucine; amino acids havingnon-aromatic, hydroxyl-containing side chains, such as serine andthreonine; amino acids having acidic side chains, such as aspartic acidand glutamic acid; amino acids having amide side chains, includingglutamine and asparagine; basic amino acids, including lysine, arginineand histidine; amino acids having aromatic ring side chains, includingphenylalanine, tyrosine and tryptophan; and amino acids havingsulfur-containing side chains, including cysteine and methionine.Additionally, amino acids having acidic side chains, such as asparticacid and glutamic acid, are considered interchangeable herein with aminoacids having amide side chains, such as asparagine and glutamine.

Accordingly, modifications to δV1-1 that are expected to result ineffective inhibition of δPKC and a concomitant increase in cerebralblood flow include the following changes to SEQ ID NO:1 shown in lowercase: tFNSYELGSL (SEQ ID NO:5), aFNSYELGSL (SEQ ID NO:6), SFNSYELGtL(SEQ ID NO:7), including any combination of these three substitutions,such as tFNSYELGtL (SEQ ID NO:8). Other potential modifications includeSyNSYELGSL (SEQ ID NO:9), SFNSfELGSL (SEQ ID NO:10), SNSYdLGSL (SEQ IDNO:11), SFNSYELpSL (SEQ ID NO:12).

Other possible modifications that are expected to produce a peptide thatfunctions in the invention include changes of one or two L to I or V,such as SFNSYEiGSv (SEQ ID NO:13), SFNSYEvGSi (SEQ ID NO:14), SFNSYELGSv(SEQ ID NO:15), SFNSYELGSi (SEQ ID NO:16), SFNSYEiGSL (SEQ ID NO:17),SFNSYEvGSL (SEQ ID NO:18), aFNSYELGSL (SEQ ID NO:19), any combination ofthe above-described modifications, and other conservative amino acidsubstitutions described herein.

Fragments and modification of fragments of δV1-1 are also contemplated,including: YELGSL (SEQ ID NO:20), YdLGSL (SEQ ID NO:21), fdLGSL (SEQ IDNO:22), YdiGSL (SEQ ID NO:23), iGSL (SEQ ID NO:24), YdvGSL (SEQ IDNO:25), YdLpsL (SEQ ID NO:26), YdLgiL (SEQ ID NO:27), YdLGSi (SEQ IDNO:28), YdLGSv (SEQ ID NO:29), LGSL (SEQ ID NO:30), iGSL (SEQ ID NO:31),vGSL (SEQ ID NO:32), LpSL (SEQ ID NO:33), LGiL (SEQ ID NO:34), LGSi (SEQID NO:35), LGSv (SEQ ID NO:36).

Accordingly, the term “a δV1-1 peptide” as used herein further refers toa peptide identified by SEQ ID NO:1 and to a peptide having an aminoacid sequence having the specified percent identity described herein tothe amino acid sequence of SEQ ID NO:1, including but not limited to thepeptides set forth in SEQ ID NOS:5-19, as well as fragments of any ofthese peptides that retain activity for increasing cerebral blood flowas described herein, as exemplified by but not limited to SEQ IDNOS:20-36.

Modifications to δV1-2 that are expected to result in effectiveinhibition of δPKC and a concomitant increase in cerebral blood flowinclude the following changes to SEQ ID NO:2 shown in lower case:ALsTDRGKTLV (SEQ ID NO:37), ALTsDRGKTLV (SEQ ID NO:38), ALTTDRGKsLV (SEQID NO:39), and any combination of these three substitutions, ALTTDRpKTLV(SEQ ID NO:40), ALTTDRGrTLV (SEQ ID NO:41), ALTTDkGKTLV (SEQ ID NO:42),ALTTDkGkTLV (SEQ ID NO:43), changes of one or two L to I, or V andchanges of V to I, or L and any combination of the above. In particular,L and V can be substituted with V, L, I R and D, E can be substitutedwith N or Q. One skilled in the art would be aware of other conservativesubstitutions that may be made to achieve other derivatives of δV1-2 inlight of the description herein.

Accordingly, the term “a δV1-2 peptide” as further used herein refers toa peptide identified by SEQ ID NO:2 and to a peptide having an aminoacid sequence having the specified percent identity described herein tothe amino acid sequence of SEQ ID NO:2, including but not limited to thepeptides set forth in SEQ ID NOS:37-43, as well as fragments of any ofthese peptides that retain activity for increasing cerebral blood flowas described herein.

Modifications to δV1-5 that are expected to result in effectiveinhibition of δPKC and a concomitant increase in cerebral blood flow asdescribed herein include the following changes to SEQ ID NO:3 shown inlower case:

rAEFWLDLQPQAKV; (SEQ ID NO: 44) KAdFWLDLQPQAKV; (SEQ ID NO: 45)KAEFWLeLQPQAKV, (SEQ ID NO: 46) KAEFWLDLQPQArV, (SEQ ID NO; 47)KAEyWLDLQPQAKV, (SEQ ID NO: 48) KAEFWiDLQPQAKV, (SEQ ID NO: 49)KAEFWvDLQPQAKV, (SEQ ID NO: 50) KAEFWLDiQPQAKV, (SEQ ID NO: 51)KAEFWLDvQPQAKV, (SEQ ID NO: 52) KAEFWLDLnPQAKV, (SEQ ID NO: 53)KAEFWLDLQPnAKV, (SEQ ID NO; 54) KAEFWLDLQPQAKi, (SEQ ID NO; 55)KAEFWLDLQPQAKI, (SEQ ID NO: 56) KAEFWaDLQPQAKV, (SEQ ID NO: 57)KAEFWLDaQPQAKV, (SEQ ID NO; 58) and KAEFWLDLQPQAKa. (SEQ ID NO: 59)

Fragments of δV1-5 are also contemplated, including:

KAEFWLD, (SEQ ID NO: 60) DLQPQAKV, (SEQ ID NO: 61) EFWLDLQP, (SEQ ID NO:62) LDLQPQA, (SEQ ID NO: 63) LQPQAKV, (SEQ ID NO: 64) AEFWLDL, (SEQ IDNO: 65) and WLDLQPQ. (SEQ ID NO: 66)

Modifications to fragments of δV1-5 are also contemplated and includethe modifications shown for the full-length fragments as well as otherconservative amino acid substitutions described herein. The term “aδV1-5 peptide” as further used herein refers to SEQ ID NO:3 and to apeptide having an amino acid sequence having the specified percentidentity described herein to an amino acid sequence of SEQ ID NO:3, aswell as fragments thereof that retain activity for increasing cerebralblood flow as described herein.

Modifications to δV5 that are expected to result in effective inhibitionof δPKC and a concomitant increase in cerebral blood flow as describedherein include making one or more conservative amino acid substitutions,including substituting: R at position 3 with Q: S at position 8 with T;F at position 15 with W; V at position 6 with L and D at position 30with E; K at position 31 with R; and E at position 53 with D, andvarious combinations of these modifications and other modifications thatcan be made by the skilled artisan in light of the description herein.

Fragments of δV5 are also contemplated, and include, for example, thefollowing:

SPRPYSNF, (SEQ ID NO: 67) ROYSNFDQ, (SEQ ID NO: 68) SNFDQEFL, (SEQ IDNO: 69) DQEFLNEK, (SEQ ID NO: 70) FLNEKARL, (SEQ ID NO: 71) LIDSMDQS,(SEQ ID NO: 72) SMDQSAFA, (SEQ ID NO: 73) DQSAFAGF; (SEQ ID NO: 74)FVNPKFEH, (SEQ ID NO: 75) KFEHLLED, (SEQ ID NO: 76) NEKARLSY, (SEQ IDNO: 77) RLSYSDKN, (SEQ ID NO: 78) SYSDKNLI, (SEQ ID NO: 79) DKNLIDSM,(SEQ ID NO: 80) PFRPKVKS, (SEQ ID NO: 81) RPKVKSPR, (SEQ ID NO: 82) andVKSPRPYS. (SEQ ID NO: 83)

Modifications to fragments of δV5 are also contemplated and include themodifications shown for the full-length fragments as well as otherconservative amino acid substitutions described herein. The term “a δV5peptide” as further used herein refers to SEQ ID NO:4 and to a peptidehaving an amino acid sequence having the specified percent identitydescribed herein to an amino acid sequence of SEQ ID NO:4, as well asfragments thereof that retain activity for increasing cerebral bloodflow. The inhibitors used for treatment herein may include a combinationof the peptides described herein.

Other suitable molecules or compounds, including small molecules, thatmay act as inhibitors of δPKC may be determined by methods known to theart. For example, such molecules may be identified by their ability toinhibit translocation of δPKC to its subcellular location. Such assaysmay utilize, for example, fluorescently-labeled enzyme and fluorescentmicroscopy to determine whether a particular compound or agent may aidin the cellular translocation of δPKC. Such assays are described, forexample, in Schechtman, D. et al., J. Biol. Chem. 279(16):15831-15840(2004) and include use of selected antibodies. Other assays to measurecellular translocation include Western blot analysis as described inDorn, G. W., II et al., Proc. Natl. Acad. Sci. U.S.A. 96(22):12798-12803(1999) and Johnson, J. A. and Mochly-Rosen, D., Circ Res. 76(4):654-63(1995).

The inhibitors may be modified by being part of a fusion protein. Thefusion protein may include a protein or peptide that functions toincrease the cellular uptake of the peptide inhibitors, has anotherdesired biological effect, such as a therapeutic effect, or may haveboth of these functions. For example, it may be desirable to conjugateor otherwise attach, the δV1-1 peptide, or other peptides describedherein, to a cytokine or other protein that elicits a desired biologicalresponse. The fusion protein may be produced by methods known to theskilled artisan. The inhibitor peptide may be bound, or otherwiseconjugated, to another peptide in a variety of ways known to the art.For example, the inhibitor peptide may be bound to a carrier peptide,such as a cell permeable carrier peptide, or other peptide describedherein via cross-linking wherein both peptides of the fusion proteinretain their activity. As a further example, the peptides may be linkedor otherwise conjugated to each other by an amide bond from theC-terminal of one peptide to the N-terminal of the other peptide. Thelinkage between the inhibitor peptide and the other member of the fusionprotein may be non-cleavable with a peptide bond, or cleavable with, forexample, an ester or other cleavable bond known to the art.

Furthermore, in other forms of the invention, the carrier protein, suchas a cell permeable carrier peptide, or other peptide that may increasecellular uptake of the peptide inhibitor may be, for example, aDrosophila Antennapedia homeodomain-derived sequence which is set forthin SEQ ID NO:84 (CRQIKIWFQNRRMKWKK), and may be attached to theinhibitor by cross-linking via an N-terminal Cys-Cys bond as discussedin Theodore, L., et al. J. Neurosci. 15:7158-7167 (1995); Johnson, J.A., et al. Circ. Res 79:1086 (1996). Alternatively, the inhibitor may bemodified by a Transactivating Regulatory Protein (Tat)-derived transportpolypeptide (such as from amino acids 47-57 of Tat shown in SEQ IDNO:85; YGRKKRRQRRR) from the Human Immunodeficiency Virus, Type 1, asdescribed in Vives, et al., J. Biol. Chem, 272:16010-16017 (1997), U.S.Pat. No. 5,804,604 and Genbank Accession No. AAT48070; or withpolyarginine as described in Mitchell, et al. J. Peptide Res. 56:318-325(2000) and Rothbard, et al., Nature Med. 6:1253-1257 (2000). Theinhibitors may be modified by other methods known to the skilled artisanin order to increase the cellular uptake of the inhibitors.

The inhibitors may be advantageously administered in various forms. Forexample, the inhibitors may be administered in tablet form forsublingual administration, in a solution or emulsion. The inhibitors mayalso be mixed with a pharmaceutically-acceptable carrier or vehicle. Thevehicle may be a liquid, suitable, for example, for parenteraladministration, including water, saline or other aqueous solution, ormay be an oil or an aerosol. The vehicle may be selected for intravenousor intraarterial administration, and may include a sterile aqueous ornon-aqueous solution that may include preservatives, bacteriostats,buffers and antioxidants known to the art. In the aerosol form, theinhibitor may be used as a powder, with properties including particlesize, morphology and surface energy known to the art for optimaldispersability. In tablet form, a solid vehicle may include, forexample, lactose, starch, carboxymethyl cellulose, dextrin, calciumphosphate, calcium carbonate, synthetic or natural calcium allocate,magnesium oxide, dry aluminum hydroxide, magnesium stearate, sodiumbicarbonate, dry yeast or a combination thereof. The tablet preferablyincludes one or more agents which aid in oral dissolution. Theinhibitors may also be administered in forms in which other similardrugs known in the art are administered.

The inhibitors utilized are capable of chronic administration withoutcausing desensitization of the patient to the inhibitor. That is, theinhibitors can be administered multiple times, or after a prolongedperiod of time including one, two or three or more days; one two, orthree or more weeks or several months to a patient and will continue tocause an increase in the flow of blood in the respective blood vessel.

The inhibitors may be administered to a patient by a variety of routes.For example, the inhibitors may be administered parenterally, includingintraperitoneally; intravenously; intraarterially; subcutaneously, orintramuscularly. The inhibitors may also be administered via a mucosalsurface, including rectally, and intravaginally; intranasally; byinhalation, either orally or intranasally; orally, includingsublingually; intraocularly and transdermally. Combinations of theseroutes of administration are also envisioned. A preferred mode ofadministration is by infusion or reperfusion through the occluded orpartially-occluded cerebral artery, or an artery that is connected tosuch an occluded or partially-occluded artery. By “partially-occludedcerebral artery” it is meant herein a cerebral artery in which bloodflow is reduced after a blood flow reducing event affecting the brainblood vessels when compared to blood flow prior to such event or attack.Such blood flow reducing events include an ischemic attack or otherhypoxic event, and vasoconstriction or vasospasm which may follow, forexample, a hemorrhagic stroke. Included in the definition of“partially-occluded cerebral artery” is a cerebral artery in which bloodflow is reduced compared to a baseline or standard blood flow rate forthat blood vessel. Such rates are known to the skilled artisan.

A therapeutically effective amount of the inhibitor is provided. As usedherein, a therapeutically effective amount of the inhibitor is thequantity of the inhibitor required to increase blood flow in a mammalianbrain blood vessel and/or to reduce the cell, tissue or organ damage ordeath that occurs due to various cell damaging events, including anischemic attack, vasoconstriction or a vasospasm, including following ahemorrhagic stroke; due to chronic high blood pressure; and/or due toidiopathic causes. This amount will vary depending on the time ofadministration (e.g., prior to an ischemic event, at the onset of theevent or thereafter), the route of administration, the duration oftreatment, the specific inhibitor used and the health of the patient asknown in the art. The skilled artisan will be able to determine theoptimum dosage. Generally, the amount of inhibitor typically utilizedmay be, for example, about 0.0005 mg/kg body weight to about 50 mg/kgbody weight, but is preferably about 0.05 mg/kg to about 0.5 mg/kg.

The amount of inhibitor is preferably sufficient to increase the flow ofblood by at least about 5%, preferably at least about 25%, further atleast about 50%, more preferably at least about 75% and further at leastabout 100% compared to the flow of blood prior to the treatment,including, for example, during the onset of an ischemic event or after aspecified time period after the onset of an aforementioned event, suchas up to about 24 hours after the onset. The blood flow is increased bythe above-recited levels typically when compared to the flow of blood atthe onset of an aforementioned event, or about 2 hours, about 3 hours orabout 6 hours after the onset of the event. It is understood thatincreases in blood flow larger than those recited above may also beobtained.

The blood vessels in which the blood flow may be increased include anyof the blood vessels of the brain in which increased blood flow isdesired. Such vessels also include those which may be totally orpartially occluded, which may be susceptible to occlusion, as well asthose vessels which have been exposed to a hypoxic event, such as anischemic stroke, or other event, such as vasoconstriction or vasospasm,which decreases delivery of cellular nutrients, including glucose. Suchvessels include, for example, cerebral arteries, such as the anteriorcerebral artery, the middle cerebral artery and the posterior cerebralartery. Brain, or other, blood vessels that supply blood to the otherbrain blood vessels may also be treated to increase the blood flow asdescribed herein. Such blood vessels include, for example, the vertebralarteries and the carotid arteries, such as the internal carotidarteries, the common carotid arteries and the external carotid arteries.Blood flow in arteries that branch from the aforementioned arteries mayalso be increased as described in the present invention. For example,the lenticulostriate arteries which branch from the middle cerebralartery, may be treated according to the methods described herein. Bloodflow may further be increased in the cerebral microvascular capillariesand arterioles, and such vessels are well known in the art. This list ofbrain blood vessels amenable for treatment to increase cerebral bloodflow therein is not exhaustive. In light of the description herein, oneskilled in the art is aware of other brain blood vessels that may betreated according to the methods described herein.

The patient to be treated is typically one in need of such treatment,including one that is susceptible to, or has experienced, a decrease incerebral blood flow from one of a number of events, such as an ischemicor hypoxic event or otherwise has the potential to incur cellular,tissue or organ damage as a result of such events. The patients caninclude those experiencing, or who are otherwise prone to experiencing,decreased cerebral blood flow due to vasoconstriction or vasospasmfollowing hemorrhagic stroke; chronic high blood pressure and/or or dueto idiopathic causes. The patient is furthermore typically a vertebrate,preferably a mammal, and including a human. Other animals which may betreated include farm animals, such as horse, sheep, cattle, and pigs.Other exemplary animals that may be treated include cats, dogs; rodents,including those from the order Rodentia, such as mice, rats, gerbils,hamsters, and guinea pigs; members of the order Lagomorpha, includingrabbits and hares, and any other mammal that may benefit from suchtreatment. The patient is preferably treated in vivo, preferably at theonset of an ischemic event. The patient may also be treated after about1 minute to about 10 hours, but preferably between about 1 minute toabout 2 hours, and further preferably after no more than about 24 hours,after occurrence of the ischemic or other event leading to hypoxiaand/or cellular nutrient deprivation.

The patient is typically one who can benefit from increased cerebralblood flow. For example, a patient who has experienced a hypoxic event,such as an ischemic stroke, or who has vasoconstriction or vasospasmfollowing hemorrhagic stroke; due to chronic high blood pressure; and/ordue to idiopathic causes may benefit from increased blood flow to theischemic penumbra as previously mentioned herein.

The ischemic penumbra, which is potentially salvageable tissue, may beidentified in a patient by methods known to the skilled artisan. Forexample, the ischemic penumbra may be identified by observingdifferences in the abnormal region defined by perfusion-weighted imagingand diffusion-weighted imaging as described in Duong, T. Q. and Fisher,M., Curr. Atheroscl. Rep. 6:267-273 (2004). This difference in thedefined regions is known as the perfusion-diffusion mismatch. Theperfusion-diffusion mismatch region is presumed to approximate theischemic penumbra. Vasoconstriction and vasospasm are usually detectedusing digital subtraction angiography as known in the art.

In yet another aspect of the invention, kits for increasing blood flowin a vertebrate, including a mammalian, brain blood vessel characterizedby, or otherwise experiencing, decreased blood flow due to a disease orcondition, including an ischemic event, vasoconstriction or vasospasmfollowing, for example, a hemorrhagic stroke; due to chronic high bloodpressure; and/or due to idiopathic causes are provided. In one form, akit includes an inhibitor of δPKC and instructions for using theinhibitor as described herein to increase blood flow in the respectivebrain blood vessel. In certain forms of the invention, the inhibitor isa δV1-1, δV1-2, δV1-5 or a δV5 peptide as described herein, or fragmentsor derivatives of these peptides as described herein, or somecombination of these peptides, their fragments and/or their derivatives.The kit may further include a syringe or other similar device known inthe art for administering the inhibitor. The components of the kit areplaced in a container, such as a box, and each component is spacedrelative to each other in the box. The inhibitors may be contained in asterilized vial or similar container. If the inhibitors are provided indried or lyophilized form and require reconstitution, the kit mayfurther include a pharmaceutically-acceptable carrier as previouslydescribed herein in a sterilized vial or similar container. A preferredcarrier is a sterile solution as described herein to dissolve theinhibitor.

Reference will now be made to specific examples illustrating theinvention described above. It is to be understood that the examples areprovided to illustrate preferred embodiments and that no limitation tothe scope of the claims is intended thereby.

EXAMPLE 1 Increased Cerebral Blood Flow in Rats Subjected toIschemic/Reperfusion Injury

The present example shows that increased blood flow was achieved in arat middle cerebral artery by treating the rat with a δV1-1 peptide.

Materials

Male Sprague-Dawley rats (280-320g; n=8) were used for cerebral ischemiaand reperfusion, and subsequent monitoring of cerebral blood flow. Tator δV1-1 peptides were synthesized and conjugated via a Cys S—S bond asdescribed previously (Chen, et al., Proc. Natl. Acad. Sci. USA25(20):11114-11119 (2001); Inagaki, et al., Circulation11(108):2304-2307 (2003)). Rats were sacrificed by overdose ofisoflurane immediately following procedure

Methods

Ischemia and reperfusion were induced in male Sprague-Dawley rats usingan occluding intraluminal suture as described in Maier, et al., J.Neurosurg. 94(1):90-96 (2001). Briefly, an uncoated 30 mm long segmentof 3-0 nylon monofilament suture with the tip rounded by a flame wasinserted into the stump of the external carotid artery and advanced intothe internal carotid artery 19-20 mm from the bifurcation to occlude theostium of the middle cerebral artery (MCA). Following placement of thesuture, the wound was closed and animals were placed back in cages for aperiod of two hours. Following this period, animals were reanestheizedand the suture was withdrawn to allow reperfusion of the brain.

Immediately following reperfusion, the rats were placed in a stereotaxicframe, and a burr hole was drilled through the skull 1 mm posterior and6 mm lateral to the bregma on the ipsilateral hemisphere correspondingto the ischemic territory. Cerebral blood flow (CBF) was measured viathis burr hole using a laser Doppler probe controlled by amicromanipulator (Laserflo BPM403A laser-Doppler flowmeter, Vasamedic,St. Paul, Minn.). The display is digital only, collects eight datapoints per second, and was set to give a moving average of data every0.1 sec. (Perez-Pinzon, et al., J. Neurolog. Sci. 153 (1):25-31 (1997);Borlongan, et al., Brain Res. 1010(1-2):108-116 (2004)). CBF readingsbegan at 30 minutes from the onset of reperfusion. Following a period of20-30 minutes to establish baseline CBF, a δPKC inhibitor, the δV1-1peptide (which was conjugated to Tat for delivery), was injected byintraperitoneal bolus, and blood flow was monitored for an additional20-30 minutes. Sham animals did not receive ischemia, but were otherwisesimilarly treated.

Results

It was found that bolus, intraperitoneal delivery of the δV1-1 peptideincreased cerebral blood flow within 30 minutes, in rats subjected toischemic/reperfusion injury as shown in FIG. 2. Sham-treated animalsshowed no change in blood flow following treatment with the δV1-1peptide within 30 minutes of delivery as seen in FIG. 1. In addition,delivery of saline alone or Tat control peptides had no effect on CBF ineither sham or ischemia treated animals (data not shown).

EXAMPLE 2 Effect of Chronic Administration of an Inhibitor of δPKC onCerebral Blood Flow in Rats Subjected to Ischemic/Reperfusion Injury

The present example shows that rats subjected to ischemic/reperfusioninjury and treated multiple times with a δV1-1 peptide did not becomedesensitized to the inhibitor. The experiment was carried out asdescribed in Example 1.

Results

Repeated intraperitoneal administrations of δV1-1 peptide furtherincreased blood flow as shown in FIG. 3.

EXAMPLE3 Microscopy Inspection of Brain Tissue in Rats Subjected toIschemic/Reperfusion Injury

The present example shows that delivery of δV1-1-TAT following strokeimproves cerebrovascular pathology. More specifically, intraperitonealdelivery of δV1-1-TAT following middle cerebral artery occlusionincreased the number of patent microvessels and improved microvascularpathology in the ischemic penumbra following stroke.

Methods

Sprague-Dawley rats underwent 120 minutes MCA occlusion, followed by 4hrs of reperfusion. Rats were treated with either TAT or δV1-1-TATpeptides at the onset of reperfusion by bolus intraperitoneal injection(0.2 mg/kg in 1 mL, n=4 per group). Sham animals were similarly treated,but did not undergo ischemia, and did not receive peptides (n=4).Following the reperfusion period, rats were heavily anesthetized usingisoflurane, and underwent transcardial perfusion using 0.7% NaCl/PBSfollowed by 4% formaldehyde in 0.7% NaCl/PBS.

Brains were removed and kept at 4° C. overnight in 2% formaldehyde/2%glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.3, Brain sections(˜1 mm³) were then dissected from the cortical surface, at ˜1 mmanterior/˜6 mm lateral to bregma on the ipsilateral cortex(corresponding to the area measured by laser Doppler flowmetry). Tissuewas post-fixed in 1% osmium tetroxide for 1 hr, washed with water, andstained in 1% uranyl acetate solution. Tissue was dehydrated using anethanol gradient followed by 100% propylene oxide, and infiltrated withEpon and Embed 812 medium, placed in molds, and polymerized overnight at65° C. before slicing. Sections (75 and 90 nm) were picked up onformvar/Carbon coated Cu grids, stained for 15 seconds in 1:1Super-saturated uranyl acetate (7.7%) in acetone followed by staining in0.2% lead citrate for 3 to 4 minutes (all reagents from ElectronMicroscopy Sciences, PA). Tissue was observed in the JEOL 1230transmission electron microscope (TEM) at 80 kV and photos were takenusing a Gatan Multiscan 791 digital camera.

To quantitate number of patent vessels, 10-15 non-overlapping fields(1200× magnification) were photographed from 2-3 slices taken from eachanimal. Vessels were identified and counted if lumen and endotheliallining was apparent; vessels were confirmed by scanning over thesefields at higher magnification. Assessments of vessel count wereperformed by an observer blinded to treatment group. The number ofvessels per field was then averaged for each group of animals (n=4 pergroup). Statistical differences between groups were analyzed using anunpaired t-test. Higher magnification images (5000-15000×) were taken toassess microvascular morphology.

Results

Defined regions of the cerebral cortex corresponding to the ischemicpenumbra (1 mm posterior/6 mm lateral to bregma) were examined. Lowermagnification images (1200×) were first used to quantitate microvesselnumber, and these images are shown in FIGS. 4A-4C. Vessels were countedif a recognizable microvascular lumen and surrounding endothelial liningwas identified. In ischemic animals treated with TAT peptide, asignificant reduction in the number of vessels was observed comparedwith δV1-1-TAT treated tissue (TAT, 1.6±0.6 vessels/field; δV1-1-TAT,3.1±0.6 vessels/field; p<0.05; n=4; FIG. 5 and FIGS. 4D-4F). The numberof microvessels in animals subject to ischemia followed with δV1-1TATtreatment at reperfusion was similar to that in sham animals (sham,3.8+/−0.4 vessels/field; n.s.; n=4; FIG. 5 and FIG. 4C vs. FIG. 4A).

Features of healthy microvascular structure were observed using highermagnification TEM in cortical tissue from sham control animals. Theseincluded intact basal lamina, clear and patent vessel lumens and lack ofabnormal perivascular spaces (FIGS. 4D, 4G, 4J). Evidence ofultrastructural damage in brain tissue from ischemic animals wasobserved (FIG. 4I, 4J), including signs of vascular edema, swollenastrocyte end-foot processes, enlarged endothelial nuclei, breakdown ofthe basal lamina, compression of vascular lumen, and luminal surfaceirregularities. However, in ischemic animals treated with δV1-1-TAT (0.2mg/kg) at reperfusion only, microvascular structure appearedsignificantly less damaged (FIGS. 4F, 4I, 4L); some astrocyte end-footswelling was observed in a small number of vessels (FIG. 4F), howevermost vessels appeared normal (FIGS. 4I, 4L).

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modificationspermutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. A method of treating an individual with a disease that causesdecreased cerebral blood flow or an idiopathic cause that causesdecreased cerebral blood flow, comprising administering atherapeutically effective amount of a peptide inhibitor of δ proteinkinase C (δPKC), whereby said administering increases blood flow in abrain blood vessel.
 2. The method according to claim 1, wherein saidpeptide is δV1-1 having an amino acid sequence set forth in SEQ ID NO:1.4. The method according to claim 1, wherein said peptide is δV1-1 havingan amino acid sequence set forth in SEQ ID NO:1, δV1-2 having an aminoacid sequence set forth in SEQ ID NO:2, δV1-5 having an amino acidsequence set forth in SEQ ID NO:3, or δV5 having an amino acid sequenceset forth in SEQ ID NO:4.
 5. The method according to claim 1, whereinsaid peptide has an amino acid sequence having at least about 95%identity to the amino acid sequence of δV1-1 set forth in SEQ ID NO:1,at least about 95% identity to the amino acid sequence of δV1-2 setforth in SEQ ID NO:2, at least about 95% identity to the amino acidsequence of δV1-5 set forth in SEQ ID NO:3, or at least about 95%identity to the amino acid sequence of δV5 set forth in SEQ ID NO:4. 6.The method according to any one of claims 1, wherein said blood vesselis a cerebral artery, capillary, or arteriole.
 7. The method accordingto claim 6, wherein said cerebral artery is an anterior cerebral artery,a middle cerebral artery or a posterior cerebral artery.
 8. The methodaccording to claim 7, wherein said peptide inhibitor is administered inan amount effective to increase blood flow at least about 10% relativeto blood flow prior to administering said medicament.
 9. The methodaccording to claim 7, wherein said peptide inhibitor is administered inan amount effective to increase blood flow at least about 50% relativeto blood flow prior to administering said inhibitor.
 10. The methodaccording to claim 9, wherein said inhibitor of δPKC is conjugated to acarrier peptide.
 11. The method according to claim 10, wherein saidcarrier peptide has the amino acid sequence set forth in SEQ ID NO:84 orSEQ ID NO:85.
 12. The method according to claim 11, wherein saidinhibitor is administered by a parenteral route.
 13. The methodaccording to claim 12, wherein said inhibitor is chronicallyadministered without causing desensitization to said inhibitor.
 14. Themethod according to claim 13, wherein said inhibitor is administeredmultiple times to a patient.
 15. The method of claim 1, wherein thedisease is hypertension or wherein the individual has a vasoconstrictionor vasospasm due to an idiopathic cause.
 16. A kit for increasing bloodflow in a mammalian brain blood vessel in an individual with a diseasethat causes decreased cerebral blood flow or an idiopathic cause thatcauses decreased blood flow, comprising a peptide inhibitor of δPKC andinstructions for using said inhibitor to increase blood flow in saidblood vessel.
 17. The kit of claim 16, wherein said peptide is δV1-1having an amino acid sequence set forth in SEQ ID NO:1.
 18. The kit ofclaim 16, wherein said peptide is δV1-1 having an amino acid sequenceset forth in SEQ ID NO:1, δV1-2 having an amino acid sequence set forthin SEQ ID NO:2, δV1-5 having an amino acid sequence set forth in SEQ IDNO:3, δV5 having an amino acid sequence set forth in SEQ ID NO:4, or acombination thereof.
 19. The kit of claim 16, wherein said peptide hasan amino acid sequence having at least about 95% identity to the aminoacid sequence of δV1-1 set forth in SEQ ID NO:1, at least about 95%identity to the amino acid sequence of δV1-2 set forth in SEQ ID NO:2,at least about 95% identity to the amino acid sequence of δV1-5 setforth in SEQ ID NO:3, or at least about 95% identity to the amino acidsequence of δV5 set forth in SEQ ID NO:4.
 20. The kit of claim 16,wherein said inhibitor of δPKC is conjugated to a carrier peptide.